This invention relates to novel compositions of matter which are inhibitors of the enzyme phospholipase A.sub.2 (PLA.sub.2) at the cell membrane, to methods for their production and to methods for their use to treat PLA.sub.2 -related abnormal conditions.
Phospholipase A.sub.2 (PLA.sub.2, EC 3.1.1.4), which hydrolyzes phospholipids at the sn-2 position to produce a fatty acid and a lysophospholipid, is present in almost all cell types and subcellular fractions. See Van den Bosch, H. (1982) "Phospholipids" (Hawthorne, N.J. & Ansell, G.D., Editors), pp. 313-357 (Elsevier Pub., Amsterdam, N.Y.) The activity of this enzyme has been correlated with various cell functions, particularly with secretory processes such as exocytosis and eicosanoid production (prostaglandins, thromboxanes and leukotrienes) See Waite M. (1987) "Phospholipases", Plenum Press, N.Y. Accordingly, PLA.sub.2 -inhibitors were proposed for treatment of diseases which are associated with enhanced cellular secretion, such as allergy (secretion of hystamine) inflammation (secretion of lysosomal enzymes and peroxides) or thrombosis (secretion of thromboxane)
It is accepted that the enzyme involved in these processes is located in the cell-surface membrane (the plasma membrane), and regulation of the membrane-enzyme's activity is required for regulation of cellular secretion. See Blackwell, G.J. and Flower, R.J. (1983). Br. Med. Bull. 39, 260-264. A number of PLA.sub.2 inhibitors have been considered for treatment of oversecretion-related pathological states. Among them are the lipocortin-like proteins which appear in mammalian tissues and assumed to be induced by glucocorticoids (although this has not been unequivocally proven). However, the prolonged administration of steroids has many side effects and, in general, is undesirable. The lipocortin-like proteins can be provided exogenously and might affect the cell membrane PLA.sub.2 activity and cellular secretion. However, since these substances are calcium-binding proteins they undesirably interfere with many other cellular functions. See: Crompton et al. (1988) Cell, 55, 1-3.
Other inhibitors, including N-derivatives of phosphotidylserine (see Martina Lagunoff (1982) Biochemistry 21, 1254-1260) are synthetic or natural products of smaller molecular weight which can be administered exogeneously. However, they are also internalized by the cell interfere with the vital lipid metabolism and are therefore cytotoxic. An extracellular inhibitor of PLA.sub.2 which affects the enzyme at the cell membrane surface but does not penetrate into the cell would, therefore, be very desirable. The PLA.sub.2 inhibitors of this invention fulfill these requirements.
In a paper published in FEBS Lett. (1986) 200 (1), pp. 165-8, which we coauthored with Nurit Reisfeld, we report the synthesis of a cell-impermeable inhibitor of phospholipase A.sub.2, by acylating the amino group of phosphatidylserine (PS) with dodecanedioic acid and linking the resulting free carboxyl group to a high molecular weight (70,000) dextran-hydrazide. This inhibitor incorporates into lipid membranes and is capable of blocking the hydrolysis of membrane phospholipids by snake venom as well as by cell membrane PLA.sub.2.
This specific PLA.sub.2 inhibitor, although cell-impermeable, has been abandoned since dextran-hydrazide is an undesirable moiety from a physiological point of view, and we have since found that linkage of PLA.sub.2 inhibiting moieties to other carrier (polymer) moieties produces cell-impermeable PLA.sub.2 -inhibitors with markedly better properties. These preparations are capable of inhibiting cellular secretion and eicosanoid production, as well as membrane-PLA.sub.2 activity in intact cells without impairing the cell viability.